The main focus of this research proposal is to elucidate the molecular machinery involved in trafficking of AMPA receptors during both developmental and activity dependent paradigms. This study will further the understanding of how pre synaptic signals can lead to acute as well was as long-term changes in synaptic efficacy. This proposal has the advantage of utilizing a wide range of techniques to address the important questions posed. Using a combination of electrophysiological, biochemical, molecular, and microscopy methods, interactions of the GluR1 AMPA receptor subunit with intracellular synaptic proteins can be implicated as key players in synaptic function. A primary neocortical culture system will be used as a model for a functional neuronal network. Overexpression of the wildtype or mutant fluorescent-tagged AMPA receptor interacting proteins SAP97/hDLG and Protein 4.1 by the Sindbis viral expression system will enable the study of these proteins with regard to their effect on synaptic transmission. The effect of chronic (>24 HR) overexpression of SAP97 and Protein 4.1 will be assessed by measuring AMPA mediated mEPSCs. Also, surface expression of GluR1 will also be measure by biochemical methods after overexpression of these proteins. Finally, using a novel assay for long-term potentiation in cultured neurons overexpressing either normal or mutant forms of SAP97 and Protein 4.1 will be employed to understand the role of these proteins in synaptic plasticity.